Amar-Costesec A, Dublet B, Beaufay H
International Institute of Cellular and Molecular Pathology, Université de Louvain, Brussels, Belgium.
Biol Cell. 1989;65(2):99-108.
Rat liver microsomes were subfractionated by isopycnic centrifugation in sucrose gradient. The subfractions were assayed for translocation and proteolytic processing of nascent polypeptides in a rabbit reticulocyte lysate programmed with total RNA from human term placenta. The distribution of the translocation and processing of prelactogen through the gradient correlated with that of the microsomal RNA (ribosomes). Microsomes became inactive upon incubation with elastase, but the proteolyzed membranes recovered their activity by recombination with the soluble and active fragment of the docking protein (SRP-receptor) from dog pancreas. When this fragment was combined with the gradient subfractions, or with the subfractions inactivated by incubation with elastase, the density profile of the translocation activity remained similar to that of RNA. Thus, its distribution cannot be accounted for merely by that of the docking protein; another membrane constituent, still unidentified, is both necessary for translocation of polypeptides and restricted to the rough portions of the endosplamic reticulum. Signal peptidase was assayed in the absence of protein synthesis, by use of preformed prelactogen and detergent-disrupted microsomes. Its density distribution was also similar to that of RNA. Several components of the endosplamic reticulum now appear to be segregated within restricted areas on either side of the membrane, and to make up a biochemically distinct domain. We propose to call it the ribosomal domain in consideration of its contribution to protein biosynthesis by bound ribosomes. This domain probably accounts for a greater part of the membrane area at the cytoplasmic than at the luminal surface, as postulated earlier to explain how enzymes of the cytoplasmic surface are relatively less abundant in the rough microsomes than those of the luminal surface [Amar-Costesec A. & Beaufay H. (1981) J. Theor. Biol. 89, 217-230].
大鼠肝脏微粒体通过在蔗糖梯度中进行等密度离心进行亚分级分离。使用来自人足月胎盘的总RNA编程的兔网织红细胞裂解物，对亚分级分离物进行新生多肽的转运和蛋白水解加工分析。催乳激素前体通过梯度的转运和加工分布与微粒体RNA（核糖体）的分布相关。微粒体与弹性蛋白酶孵育后失活，但经蛋白水解的膜通过与来自犬胰腺的对接蛋白（信号识别颗粒受体）的可溶性活性片段重组而恢复其活性。当该片段与梯度亚分级分离物或与用弹性蛋白酶孵育而失活的亚分级分离物结合时，转运活性的密度分布仍与RNA的相似。因此，其分布不能仅由对接蛋白的分布来解释；另一种仍未鉴定的膜成分对于多肽的转运是必需的，并且仅限于内质网的粗糙部分。在无蛋白质合成的情况下，使用预先形成的催乳激素前体和去污剂破坏的微粒体来测定信号肽酶。其密度分布也与RNA的相似。内质网的几个成分现在似乎在膜两侧的受限区域内分离，并构成一个生化上不同的结构域。考虑到其对结合核糖体进行蛋白质生物合成的贡献，我们建议将其称为核糖体结构域。如先前假设的那样，该结构域可能在细胞质表面比在腔表面占更大比例的膜面积，以解释为什么细胞质表面的酶在粗糙微粒体中比腔表面的酶相对较少[阿玛尔 - 科斯特塞克A.和博法伊H.（1981年）《理论生物学杂志》89，217 - 230]。
